1. Field of the Invention
This invention relates to methods and apparatus for determining distributions of radioactive materials. More particularly, it relates to methods and apparatus for determining distributions of radioactive materials wherein the distribution has been produced by a chromatography process, such as, an electrochromatography (electrophoresis) process.
2. Description of the Prior Art
By way of background, a variety of radioisotopes are currently used in chemical and medical research. Chromatographic procedures are routinely applied to separate isolate, and identify radioactively-labelled organic substances. The ability to effect such separation and identification of such radioactively-labelled materials has helped to increase their applications, especially in biochemical research. Thin-layer chromatography, column chromatographic techniques, electrophoresis, and paper chromatography can be used in suitable situations and have various sensitivities. Long-lived radioisotopes, which are generally weak beta emitters, are preferentially used for chemical and biological studies. Of particular importance are radiocarbon, C.sup.14, and radioactive hydrogen (Tritium), H.sup.3 Radioactive phosphorus, P.sup.32, radioactive sulphur, S.sup.35, and radioactive iodine, I.sup.131, are also frequently used. Autoradiograms are limited in that no direct quantitative measurement of the relative strength of different bands is provided and that the procedures are often difficult to perform and require extended periods of time.
There are two major methods of detecting radiation in electro or other chromatograms: (i) autoradiography; and (ii) counting tubes and scintillation counters. Autoradiograms are produced as a result of the ability of alpha, beta, and gamma rays to darken photographic emulsions. The dry chromatogram is pressed against an X-ray film, wrapped, and kept in the dark for a period of time depending upon the nature of the radio isotopes involved. For example, C.sup.14 labelled compounds are usually left in contact with X-ray film for 1-8 days; tritiated substances have to be exposed for more than a week; and P.sup.32 or I.sup.131 compounds can produce good autoradiograms in some cases in less than six hours. In the case of the detection of compounds containing different radioisotopes and double labelled substances on the same chromatogram, it is necessary to produce several autoradiograms over several months (i.e., the earlier autoradiographs would show spots caused by both isotopes, whereas the later autoradiographs would only show the isotope with the longer half life). Double emulsion radioautography, or mixed radioautography radiofluography can be used to detect dual labelled substances.
Paper chromatograms of radioactive substances can be quantitatively evaluated by a Geiger-Mueller counting tube, by use of a proportional counter, or by scintillation counting. Commercial devices for the detection and recording of material on an automatically transported paper strip or on a two-dimensional chromatogram are commercially available. Similar instruments for use with plates or gels are known.
Various patents have issued concerned with methods and devices for the detection of radioactively-labelled materials which have been chromatographically separated. Examples of such patents are: U.S. Pat. Nos. 3,027,453; 3,033,986; 4,019,057; 4,110,615; 4,311,908; 4,431,921; 4,028,549; 4,298,796; 3,814,939; 4,214,161; 4,267,451; 4,275,300; and 4,469,601. These publications are incorporated herein by way of reference.
In the field of biochemistry with particular reference to gene analysis, the standard technique for analyzing mixtures of polynucleotides involves the use of electrochromatography in combination with radioactive polynucleotide probes having known sequences. See E. Southern, "Gel Electrophoresis of Restriction Fragments," Methods in Enzymology, Vol. 68, 1979, pages 152-176.
In accordance with this technique, the mixture of polynucleotides to be analyzed is applied to a suitable electrophoresis gel, such as, an agarose or polyacrylamide gel, and an electric field is applied across the gel to separate the mixture into discrete bands. As a result of the separation, the polynucleotides in any particular band will in general have the same molecular weight and electrical charge.
Once the separation has been completed, denaturation is generally effected and a radioactive probe is applied to the gel and allowed to hybridize with the bands. The gel is then washed to remove unhybridized probe. As a result of this procedure, those bands, and presumably only those bands, which include polynucleotide sequences complementary to the probe sequence end up being radioactive. Radioactive bands can be obtained in other ways. For example, the mixture of polynucleotides to be analyzed can be radiolabeled, separated using electrophoresis, and then hybridized with cold probe. Non-hybridized sequences can then be removed using an enzyme, such as, mung bean nuclease or nuclease S.sub.1, which preferentially breaks down single stranded polynucleotides. Again, the final product is a gel having discrete bands of radioactivity.)
Once the hybridization has been completed, the gels must be analyzed to determine where the radioactive bands are located. It is to this aspect of the overall process that the present invention is directed.
Prior to the present invention, the location of the radioactive bands has in most cases been determined through the use of X-ray films (autoradiography). Specifically, in the standard "Southern Blot" procedure, see E. Southern, Methods in Enzymology, supra, the radioactive bands in the gel have been transferred to a second medium, such as a sheet of cellulose nitrate paper, and a sheet of photographic film has been placed next to the second medium so that the radiation emitted by the radioactive bands ban locally expose the film. The film has then been developed using conventional techniques, and the bands analyzed visually to determine the presence or absence of specific polynucleotide sequences in the mixture of polynucleotides.
This photographic procedure has numerous disadvantages which are well known in the art. For example, it can often take exceedingly long periods of time to expose the film, e.g., periods of time on the order of week or more. Moreover, the process involves numerous manipulative steps both in terms of exposing the film and in terms of developing it. Overriding these physical problems is the fact that the ultimate output of the photographic process is simply a piece of film having a series of exposed bands therein. Output of this type has in general only been interpretable by skilled personnel.
In addition to the use of autoradiography, electrophorsis gels having radioactive bands have also been analyzed by scintillation counting. In this procedure the gel containing radioactive bands sliced or cut, dissolved in a certain scintillant, and subsequent light emissions are then measured. This procedure can produce quantitative results, but resolution is limited by the size of the slices of gel (typically 1 mm or greater).
Although one of the major applications of the invention herein described involves the analysis of genetic material, chromatography employing radioactive materials is also widely used in diverse chemical, and particularly biochemical, disciplines. As with gene analysis, in these fields radioactive materials become distributed in a supporting medium and there is a need to determine the distribution of those materials within the medium. Accordingly, the present invention is applicable to these fields also.